P858: EPIGENETIC REGULATION OF CD155 ON MULTIPLE MYELOMA CELLS INFLUENCES ANTI-TUMOR T-CELL CYTOTOXICITY
Authors: L. Martinez-Verbo, P. Llinàs-Arias, L. Villanueva, C. A. García-Prieto, D. Piñeyro, G. Ferrer, M. Esteller
Journal: HemaSphere (2022)

Background: DNA methylation remains as one of the key molecular players regulating gene expression. In multiple myeloma (MM) and thanks to data available, we identified CD155 as a putative epigenetically regulated gene. Cytotoxic T-cells (CD8+) are crucial in the clearance of malignant cells and their activation is tightly regulated by immune checkpoint events (IC). Tumor cells take advantage of this system and escape immune recognition, avoiding T-cell attacks by inducing exhaustion. CD155 is involved in the CD8+ T-cell activation IC and it is of interest because could act as an inhibitory or stimulatory signal. We investigated the role of CD155 in MM and its importance in T-cell inhibition in-vitro, in relation to its epigenetic state and expression. Aims: To evaluate the role of CD155 methylation in MM cells and how it affects T-cell activation and tumor elimination. Methods: The methylation status of the promoter region of CD155 was studied in several MM cell lines from COSMIC database and experimentally validated in four (RPMI-8226, MM.1S, AMO-1 and KMS-12-BM). RPMI-8226 was selected as an unmethylated cell line for CD155 knock down model (depleted expression) and scramble model by lentiviral transduction. We analyzed if CD155 has an impact in cell proliferation and cell cycle with MTT assay and flow cytometry. Co-culture experiments were performed with the CD155 models and T-cells isolated from healthy donors for 48 hours and studied T-cell cytotoxicity by analyzing the remaining MM cells with flow cytometry. Finally, we analyzed CD155 expression and survival in MM from CoMMpass project public data. Results: We validated the in-silico methylation data by bisulfite sequencing and negative regulation by Western Blot and quantitative PCR in four different cell lines. Depletion of CD155 did not have a significant impact on cell growth, apoptosis or cell cycle. For instance, when we co-cultured the CD155 models with pre-activated T-cells, we detected more cytotoxicity towards CD155 depleted cells, whereas CD155 expressing cells resisted T-cell activity (p=0.02). In order to determine if the inhibition was mediated by interaction CD155-TIGIT, we added 10ug/ml of neutralizing αTIGIT and/or αPD1 antibody to the co-culture systems. While T-cells co-cultured with CD155 depleted cells in the presence of αTIGIT did not change their levels of cytotoxicity, expressing cells were eliminated more successfully. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and antibody combination showed a synergic effect in expressing models’ co-culture whereas CD155 depleted levels remained independent of αTIGIT presence. Supporting these results, the data obtained from the CoMMpass project showed the MM patients (N= 793) with higher expression of CD155 had shorter overall survival than lower expressing ones (p<0.001). Summary/Conclusion: In MM cells, the expression of CD155 is regulated by the methylation status of its promoter region. Unmethylated MM cells expressing CD155 promote T-cell inhibition and its depletion resulted in an improvement of T-cell cytotoxicity activity against malignant cells. The addition of anti-TIGIT and anti-PD1 validated that the CD155 T-cell inhibition is mediated by the interaction with TIGIT, independently of PD1. These findings indicate that CD155 unmethylated MM cases are at higher risk warranting further investigation.